White nylon material was chosen to make cylindrical tissue phantoms for development of bioluminescence tomography techniques. A low-level light source, delivered through an optic fiber of core diameter 200 μm, was placed at different locations on one phantom surface. The light travels through the phantom, reaches the external surface, and is captured by a liquid nitrogen-cooled CCD camera. The scattering, absorption, and anisotropy parameters of the phantom are obtained by matching the measured light transmission profiles to the profiles generated by the TracePro software. The perturbation analysis, with the homogeneous phantoms, demonstrated that the imaging system is sufficiently sensitive to capture intensity change of higher than 0.5nW/cm2 or a location shift of the light source of more than 200 microns. It is observed that the system can distinguish two point light sources with separation of about 2 mm. The perturbation analysis is also performed with the heterogeneous phantom. Based on our data, we conclude that there is inherent tomographic information in bioluminescent measures taken on the external surface of the mouse, which suggests the feasibility of bioluminescence tomography for biomedical research using the small animals, especially the mice.